U.S. patent application number 13/032443 was filed with the patent office on 2011-07-21 for on-board control for analytical elements.
This patent application is currently assigned to ROCHE DIAGNOSTICS OPERATIONS, INC.. Invention is credited to Joerg Dreibholz, Joachim Hoenes, Carina Horn, Holger Kotzan, Mihail-Onoriu Lungu, Michael Marquant, Christine Nortmeyer, Volker Unkrig.
Application Number | 20110177605 13/032443 |
Document ID | / |
Family ID | 34306334 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110177605 |
Kind Code |
A1 |
Unkrig; Volker ; et
al. |
July 21, 2011 |
On-Board Control for Analytical Elements
Abstract
The invention concerns a reagent system for the so-called
on-board control of analytical elements, in particular test strips,
containing an organic N-oxide or a nitroso compound. The invention
also concerns analytical elements containing a reagent system for a
detection reaction and a reagent system for an on-board control.
Furthermore, the invention concerns a method for checking
analytical elements in which a reagent system for an on-board
control is examined optically or electrochemically with the aid of
a measuring instrument for changes which could indicate a stress of
the analytical element.
Inventors: |
Unkrig; Volker; (Ladenburg,
DE) ; Nortmeyer; Christine; (Mannheim, DE) ;
Horn; Carina; (Biblis, DE) ; Marquant; Michael;
(Mannheim, DE) ; Lungu; Mihail-Onoriu;
(Schwegenheim, DE) ; Hoenes; Joachim;
(Zwingenberg, DE) ; Kotzan; Holger; (Ladenburg,
DE) ; Dreibholz; Joerg; (Altrip, DE) |
Assignee: |
ROCHE DIAGNOSTICS OPERATIONS,
INC.
Indianapolis
IN
|
Family ID: |
34306334 |
Appl. No.: |
13/032443 |
Filed: |
February 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10962796 |
Oct 12, 2004 |
7914752 |
|
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13032443 |
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Current U.S.
Class: |
436/106 ;
204/400; 205/775; 422/68.1; 422/73; 422/82.05 |
Current CPC
Class: |
Y10T 436/170769
20150115; G01N 33/52 20130101; Y10T 436/179228 20150115; Y10T
436/17 20150115; G01N 33/86 20130101; G01N 31/22 20130101; Y10T
436/144444 20150115; Y10T 436/104998 20150115; G01N 33/54386
20130101 |
Class at
Publication: |
436/106 ;
422/68.1; 422/82.05; 422/73; 204/400; 205/775 |
International
Class: |
G01N 21/00 20060101
G01N021/00; G01N 27/28 20060101 G01N027/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 2003 |
DE |
103 46 863.3 |
Claims
1. An analytical element comprising: a first reagent system for
detecting an analyte, and an on-board control comprising a second
reagent system that produces a signal independent of the presence
of the analyte and comprises an organic N-oxide or a nitroso
compound and a reducing agent.
2. The analytical element of claim 1, wherein said second reagent
system determines whether at least one of the analytical element,
the first reagent system and the second reagent system has been
stressed under conditions that could damage the analytical element
or affect results obtained therefrom.
3. The analytical element of claim 1, wherein the signal produced
by the second reagent system can be detected optically or
electrochemically.
4. The analytical element of claim 1, wherein the reducing agent is
glycine or glucose.
5. The analytical element of claim 1, wherein the N-oxide is
resazurin.
6. The analytical element of claim 1, wherein the nitroso compound
is a substituted p-nitrosoaniline.
7. The analytical element of claim 1, wherein the second reagent
system is integrated into the first reagent system.
8. The analytical element of claim 1, wherein the first reagent
system comprises reagents for determining coagulation
parameters.
9. The analytical element of claim 1, further comprising a
heteropoly acid in combination with said nitroso compound.
10. A method for the control of analytical elements as claimed in
claim 1, comprising detecting the signal from the second reagent
system optically or electrochemically with the aid of a measuring
instrument, wherein the signal indicates a stress of the analytical
element.
11. The method of claim 10, wherein the analytical element for
which a stress has been detected is not released by the measuring
instrument for measurement of a sample liquid with the aid of the
first reagent system.
12. The method of claim 10, wherein the signal produced by the
second regent system and a signal produced by the first reagent
system are detected optically.
13. The method of claim 10, wherein the signal produced by the
second regent system and a signal produced by the first reagent
system are detected electrochemically.
14. The method of claim 10, wherein the signal produced by the
second regent system is detected optically and a signal produced by
the first regent system is detected electrochemically.
15. The method of claim 10, wherein the signal produced by the
second regent system is detected electrochemically and a signal
produced by the first regent system is detected optically.
Description
BACKGROUND OF THE INVENTION
[0001] The invention concerns a control reagent system for an
analytical element, for example in the form of a test strip and, in
particular, a test strip for determining a coagulation parameter
which allows a differentiation between functioning analytical
elements and non-functioning analytical elements. The invention
also concerns corresponding analytical elements and methods for
their control.
[0002] So-called carrier-bound tests are being used to an
increasing extent for the qualitative and quantitative analysis of
components of a liquid sample in particular a body fluid from
humans or animals. Analytical elements (also referred to as test
elements) are used for this where at least one reagent is embedded
in a test field consisting of one or more layers, which is brought
into contact with the sample. The reaction of sample and reagent
results in a change in the analytical element that can be evaluated
visually or with the aid of an instrument (usually by reflection
photometry or electro-chemically). After a test has been carried
out the used analytical element is disposed of.
[0003] Numerous different types of analytical element are known
which differ in their measuring principle (e.g., optical or
electrochemical) and the reagents that are used and in their
construction and in particular with regard to the arrangement and
attachment of the test layers. Strip-shaped analytical elements are
of particular practical importance. These analytical elements that
are also referred to as test strips are essentially composed of an
elongate support layer made of a plastic material on which one or
more test fields are attached.
[0004] The analytical elements are packaged in primary packaging in
the interior of which they are stored until use i.e., until they
are removed by the user and a test has been carried out with
subsequent disposal. The analytical elements may be packaged
individually in their own primary packaging. Analytical element
packaging units are commonly used in which a plurality of
analytical elements are located in the interior of a common primary
packaging. The primary packaging usually contains a desiccant.
[0005] The interior of the primary packaging is usually
substantially hermetically sealed. Hence, the storage conditions
are essentially determined by the environmental conditions in the
primary packaging during storage.
[0006] Many analytical elements contain reagents which can be
damaged by certain storage conditions e.g., temperature, humidity,
oxygen, light, etc., which makes them unusable for carrying out a
reliable test. Hence, in order to avoid damage it is necessary to
store the analytical element packaging unit under certain
appropriate conditions recommended by the manufacturer. On the part
of the manufacturer, the storage life of the analytical element is
guaranteed for a certain period when stored properly.
[0007] The use of an analytical element with a damaged reagent can
lead to a false test result which may result in a serious
misinterpretation of, for example, the state of health of a person.
Hence, in the past, various attempts have been made to reduce the
risk of using analytical elements with storage damage.
[0008] For example, test reagents have been developed that are
relatively insensitive to external effects. The aim of another
development is to use elaborate primary packaging to minimize
external effects on the reagents of the analytical elements. Both
solutions are associated with substantially increased manufacturing
costs. For safety reasons a relatively short shelf life is stated.
As a result, analytical elements can no longer be used after the
shelf life date has expired although it is not possible to check
whether in fact there have been conditions which could have
resulted in damage to the reagents.
[0009] Test strips for diagnostic blood examinations are subject to
an extensive quality control before sale. Suitable shipping and
storage conditions are intensively examined before they are
launched on the market and are, for example, described on the
packaging or in the package insert. Nevertheless, it cannot be
completely excluded that strips are damaged before the expiration
date during transport of the goods to the customer or due to
incorrect storage by the customer and that there is a risk that
false measurements are obtained when they are used.
[0010] Improper transport and/or storage conditions can be
discovered by measurements using liquid controls which are
distributed as additional system components besides the instrument
and strips for most so-called point of care systems (PoC systems)
that are used decentrally (i.e., outside of special laboratories
i.e., for example, in doctor's offices, pharmacies or by the
patient at home). Disadvantages of using liquid controls for PoC
systems (e.g., for coagulation measurement systems) are their
somewhat complicated handling, the costs for using usually two test
strips and two control liquids (level 1 & level 2 control) and
the fact that although usually strips from the same production lot
and packaging are measured with the controls they are, however,
inevitably other strips than those with which the blood sample of a
patient is in fact examined.
[0011] These disadvantages are avoided by an on-board control
(abbreviated as OBC in the following) which is integrated into each
test strip and does not require an additional test liquid. Systems
with on-board controls require no control liquids but work with the
same sample liquid from which the parameter to be determined is
determined with the measurement system.
[0012] A common feature of systems with on-board controls known in
the art is that a blood sample is taken up into an analytical
element through a capillary channel and is transported by capillary
forces to a site of examination within the analytical element. The
sample is divided by one or more branches of the capillary system
and conveyed into one or more side channels. Reagents are then
located in these side channels which constitute the actual OBC.
[0013] A common disadvantage of these on-board controls is that the
test strips--in addition to the actual measurement channel for the
patient sample--require several (usually two) additional channels.
After being filled with the same patient blood, measurements are
carried out in these additional channels which should give
information about the integrity of the strip. This concept results
in high manufacturing costs since the individual channels have to
be separately provided with different reagents. Furthermore, such
control systems require comparatively large sample volumes since,
in addition to the actual measurement channel, at least one and
usually even several control channels have to be filled with
sample. Large sample volumes are regarded as being a particular
disadvantage where patients themselves have to regularly obtain the
sample i.e., for example, in so-called home monitoring especially
in the case of diabetics or patients which have to monitor their
own coagulation values since the collection of blood samples by
puncturing the skin is painful and even more painful the more blood
sample is required. Moreover, multichannel on-board control systems
suffer from difficult filling mechanisms since the sample has to
automatically penetrate into several channels and fill them.
SUMMARY OF THE INVENTION
[0014] It is against the above background that the present
invention provides certain unobvious advantages and advancements
over the prior art. In particular, the inventors have recognized a
need for improvements in on-board control systems for analytical
elements.
[0015] Although the present invention is not limited to specific
advantages or functionality, it is noted that the present invention
provides an on-board control system which does not have the
disadvantages noted above, especially with regard to manufacturing
costs and sample volume, but nevertheless reliably indicates the
potential unusability of test elements, especially as a result of
improper storage or transport conditions. In particular, damaging
humidity and/or temperature stress on the test elements should be
detected with the aid of the OBC according to the present
invention. Furthermore, the intended OBC should not react too early
in order to enable a long storage of the product at room
temperature and should enable a good discrimination between intact
and defective strips with a high precision.
[0016] In accordance with one embodiment of the present invention,
a reagent system for the on-board control of analytical elements is
provided comprising an organic N-oxide or a nitroso compound.
[0017] In accordance with another embodiment of the present
invention, an analytical element comprising a reagent system for a
detection reaction and the reagent system for the on-board control
of analytical elements according to the first embodiment of the
present invention is provided.
[0018] In accordance with still another embodiment of the present
invention, a method for the control of analytical elements is
provided comprising examining the reagent system for the on-board
control optically or electrochemically with the aid of a measuring
instrument for changes which can indicate a stress of the
analytical element.
[0019] These and other features and advantages of the present
invention will be more fully understood from the following detailed
description of the invention taken together with the accompanying
claims. It is noted that the scope of the claims is defined by the
recitations therein and not by the specific discussion of features
and advantages set forth in the present description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The following detailed description of the embodiments of the
present invention can be best understood when read in conjunction
with the following drawings, where like structure is indicated with
like reference numerals and in which:
[0021] FIG. 1 shows the structural formulae of resazurin 1 which is
converted by reduction into resorufin 2 and by further reduction
into dihydro-resorufin 3;
[0022] FIG. 2 shows absorption spectra (relative absorption A
plotted versus the wavelength .lamda. (in nm)) of resazurin 1 and
resorufin 2;
[0023] FIG. 3 shows the reflectance spectra (relative reflectance R
(in %) plotted versus the wavelength .lamda. (in nm)) of the
reagent fields of test elements that have been stressed to
different extents (1 unstressed, 2 slightly stressed, 3 strongly
stressed);
[0024] FIG. 4 shows the increase in degradation product (resorufin)
in a resazurin-containing reagent film based on the increase of the
relative reflectance R (in %) (measured at a wavelength of 620 nm)
over the stress time t (in h); and
[0025] FIG. 5 shows the change of the signal that can be measured
electrochemically during a first measuring phase lasting for 3
seconds at -700 mV versus Ag/AgCl (Q.sub.700) (lower two curves)
and a second measuring phase lasting for 1.5 seconds at -100 mV
versus Ag/AgCl (Q.sub.100) (upper two curves) on the basis of the
dependence of the measured charge Q on the stress time t (in
h).
DETAILED DESCRIPTION OF THE INVENTION
[0026] The concept according to the present invention, which can
also be referred to as a 1-channel on-board control, provides that
a reagent zone on a test element which can also be used for the
actual measurement of the patient sample, contains a substance
which changes when stressed. The change in this substance can
result in a degradation product which can either be detected
visually or by the instrument provided to measure the test strip. A
strip that has been recognized to be defective can for example not
be released by the measuring instrument for measuring the patient
sample.
[0027] The reagent system according to the present invention is
suitable for the on-board control of analytical elements. The
reagent system contains at least one chemical substance, but
typically a mixture of chemical substances. At least one of these
substances is suitable for directly or indirectly indicating
environmental conditions which could impair the reliability of an
analytical element. This property is referred to as an on-board
control property. For this purpose, damaging environmental effects
such as temperature, humidity, light, oxygen, etc., are utilized to
carry out a typically irreversible change in the said substance or
with the said substance which enables a subsequent detection of the
damaging effects. In this connection it is usually irrelevant to
determine the exact type of damaging effects; it is usually
sufficient to establish the fact that a damaging effect has taken
place.
[0028] The reagent system for on-board control can contain other
auxiliary substances in addition to the substance which has the
actual on-board control function. These may be buffer substances,
fillers, film formers and such like which are known to a person
skilled in the art in numerous embodiments in connection with
reagent formulations for analytical test elements.
[0029] In the sense of the invention analytical elements (also
referred to as test elements, analytical test elements, test
strips, test chips, test devices) are typically suitable for
determining analytes or other parameters in liquid samples and more
typically in liquid samples of human origin such as blood, serum,
plasma, urine and such like. Analytical elements in the sense of
the invention typically contain a reagent or reagent system on a
support material which generates a detectable signal that is
dependent on the analyte to be examined in the sample or on the
property of the sample to be examined. Such analytical elements are
known to a person skilled in the art in numerous embodiments.
Examples are optical or electrochemical test strips for detecting
metabolites in blood or sample liquids derived therefrom and in
particular for determining glucose, cholesterol and such like.
Furthermore, test strips are known which can be used to determine
coagulation parameters in a blood sample.
[0030] The reagent system that is located on the analytical element
and which is responsible for a detectable detection signal for an
analyte in the sample or for a property of the sample can typically
result in an optically detectable and evaluable measurement signal
or a signal that can be detected by electrochemistry. Both variants
are known to a person skilled in the art in numerous
embodiments.
[0031] The reagent system for on-board control according to the
invention is typically integrated into the reagent system for the
detection reaction. However, it is also conceivable and possible to
accommodate reagent systems for the detection reaction and reagent
systems for the on-board control in spatially separated zones on an
analytical element.
[0032] For the typical case that the reagent system for the
on-board control is integrated into the reagent system for the
detection reaction care must be taken that the reagents do not
negatively affect one another.
[0033] The reagent system for the on-board control and the reagent
system for the detection reaction can be based on the same or on
different detection principles. Thus, according to the present
invention, it is possible to optically or electrochemically
evaluate the on-board control. It is also possible to optically or
electrochemically detect the actual detection reaction. It is more
typical to electrochemically detect the on-board control as well as
the detection reaction. In this connection an optical detection can
be carried out visually or by means of an apparatus with the aid of
a photometer in which case any methods familiar to a person skilled
in the art such as reflection measurement, absorption measurement,
transmission measurement, luminescence measurement and such like
can be used. In turn, for the electrochemical detection methods
such as potentiometry, amperometry, coulometry, chrono-amperometry
and such like are suitable.
[0034] In particular, an optical change in the on-board control
field or detection reagent field can be evaluated with the naked
eye without using a measuring instrument. However, it is typical to
evaluate the on-board control and the detection field with the aid
of a measuring instrument, for example, a photometer or an
electrochemical measuring instrument. It is also possible to
evaluate the on-board control without a measuring instrument but to
evaluate the detection reaction with a measuring instrument.
[0035] The reagent formulations for the on-board control and the
detection field can be applied to a support of an analytical
element using any methods known to a person skilled in the art. In
this connection the various reagents can be applied using the same
or different methods. Some of the possible methods are for example:
application in a liquid form and subsequent drying on or into a
support; application as a coating mass by knife coating, slot
nozzle coating and the like; and printing processes such as ink-jet
printing, screen printing, dispersing, etc. It is also possible to
mount reagents already applied to a first support together with
this first support on the actual second support of the analytical
element and to join them e.g., by gluing, welding, etc.
[0036] The on-board control according to the present invention
should be able to reliably detect stressed test elements. In this
connection stress is understood to mean that test elements have
been exposed to environmental conditions which could lead to damage
or impairment of the reagents for the actual detection reaction. In
the case of analytical elements which are intended to be stored at
room temperature in closed cans provided with desiccants or
provided with moisture-impermeable foils, stress is, for example,
understood as exposure to high temperatures in the closed cans or
foils (e.g., during transport to the customer or behind a pane of
glass in sunlight) or damp storage (in the case of improperly
closed cans after removing test strips or defective foil
packaging). Of course, combinations of stress events such as
concurrent humidity or elevated temperature can result in damage.
In this case it is only important that a stress finally results in
an impairment or failure of the detection reagent system. These can
indeed be different specific conditions for the various analytical
elements. A person skilled in the art knows how he can identify
such damaging events for the respective reagent system.
[0037] According to the present invention, a stress of the
analytical element or of the reagent system for the on-board
control results in a--typically irreversible--change in the reagent
system of the on-board control. This ensures that damaging
environmental effects are also detected when the environmental
conditions have in the meantime changed into favourable conditions.
For example, a single damaging temperature elevation or a single
brief effect of humidity which has resulted in damage can thus be
reliably identified.
[0038] Surprisingly, it was found that reagents which contain
N-oxides or nitroso compounds, in particular, in combination with
reducing agents such as sugars, polyalcohols, cysteine-containing
proteins, glycine, etc., can be reduced under the same conditions
and environmental effects under which an OBC should indicate
undesired effects and negative changes of a test strip. The
degradation products formed by reduction from the N-oxides or
nitroso compounds can be detected in the test strip by suitable,
typically electrochemical or optical methods. In particular, if
sugars are used as the reducing agents, the redox properties and
hence the kinetics of the redox reaction can be adjusted by means
of the pH of the reagent.
[0039] According to the present invention, the N-oxide resazurin
can be a particularly suitable molecule for the described indicator
reaction and is thus typical.
[0040] The blue resazurin is reduced to the red resorufin (cf.,
FIG. 1) when stressed under conditions that could damage a test
strip (e.g., elevated temperatures, humidity and light) where the
reduction takes place especially in the presence of a suitable
redox partner, typically those from the reagent formulation. The
change, i.e., the decrease in the resazurin concentration with a
simultaneous increase in the resorufin concentration can be
determined visually by an optical detector in a measuring
instrument or by an electrochemical sensor.
[0041] In the case of resazurin, glycine can be a very specific
reducing agent. A combination of suitable amounts of resazurin and
glycine in an OBC reagent is more typical, in accordance with the
present invention. A minimum concentration of about 0.01 g/l
resazurin is typical, since detection of resazurin is practically
very difficult below this concentration. The maximum amount of
resazurin should not exceed about 20 mmol/l since otherwise
solubility problems can occur. As described above, glycine is not
absolutely necessary for the function of the OBC containing
resazurin; a concentration of about 250 g/l has proven to be the
maximum amount of glycine since solubility problems occur above
this amount and glycine may crystallize from the solution which in
turn can cause problems when coating the reagent mass and may lead
to inhomogeneities in the coated mass.
[0042] The remaining resazurin and/or the resorufin that is formed
can be quantified for the electrochemical detection of the OBC
reaction.
[0043] A quantification of resazurin is typical for large changes
in concentration and can for example be carried out by
electrochemical reduction of the resazurin at a potential of about
-700 mV against Ag/AgCl. At this potential the resorufin that is
formed is reduced further to dihydro-resorufin (cf., FIG. 1).
[0044] If mainly resazurin is present in the OBC reagent (and hence
the reagent is not stressed or hardly stressed) a 4-electron
transition is provoked which results in a higher current than when
mainly resorufin is present whose reduction only leads to a
conversion of 2 electrons. The current or charge measured at a
predetermined potential and in particular at a typical potential of
about -700 mV against Ag/AgCl thus allows deductions to be made
about the extent of the temperature and/or humidity stress of the
reagent.
[0045] Resorufin cannot be quantified by electrochemical
(re)oxidation of resorufin to resazurin because the reduction of
resazurin to resorufin is irreversible. A reductive detection in
the described OBC system only requires that the reduction of
resorufin is specifically detected but not the reduction of
resazurin which may be present in the reagent. This can, for
example, be accomplished by using a specific reduction potential
which is typically in the range of about -450 to about -550 mV
against Ag/AgCl.
[0046] The quantification of resorufin in the described OBC system
can be achieved especially well when in a first step the resorufin
present in the test strip is converted electrochemically into
dihydro-resorufin (referred to as OBC-prepare) and the in situ
generated dihydro-resorufin is electrochemically oxidized back to
resorufin in a second step (referred to as OBC test in the
following). This oxidation reaction typically runs at a potential
of about -100 mV against Ag/AgCl.
[0047] The intensity and specificity of the OBC test signal can be
controlled by the length of the OBC prepare phase.
[0048] In addition to the typical resazurin/resorufin system of the
present invention, nitroso compounds and, in particular,
p-nitrosoanilines are another typical example for a class of
substances which can be converted into products which can indicate
a possible damage of the test strip under conditions that can
damage test strips. p-Nitrosoanilines can, for example, be reduced
under conditions which can also damage reagents in a detection
reagent. The products (such as phenylenediamines) produced by
reduction can also be detected optically or electrochemically.
Other nitroso compounds which can be used according to the present
invention are described in the following U.S. patents: U.S. Pat.
No. 5,206,147, U.S. Pat. No. 5,334,508, U.S. Pat. No. 5,122,244 and
U.S. Pat. No. 5,286,362, the disclosures of which are incorporated
herein by reference for their teaching of nitroso compounds. A
combination of the nitroso compounds disclosed in these US patents
with heteropoly acids, in particular heteropoly acids in a
precipitated form according to U.S. Pat. No. 5,240,860, the
disclosure of which is incorporated herein by reference, which when
stressed formed readily visible heteropoly blue in the presence of
reducing agents is typical.
[0049] The invention is characterized in more detail by the
following examples which describe the advantages and properties of
the OBC according to the invention using test strips for
coagulation measurements as an example (prothrombin time test or PT
test). It is clear to a person skilled in the art that the
statements made on the basis of the coagulation test strip example
also apply to other types of test strip and in particular to those
used for the optical or electrochemical determination of blood
glucose, lipids such as cholesterol and HDL cholesterol,
triglycerides, etc., for the determination of other coagulation
parameters than PT such as aPTT, ACT, ECT, anti-factor Xa tests and
also for immunological test elements, in particular chromatography
test strips that can be evaluated optically and can be applied to
them. Accordingly, in order that the invention may be more readily
understood, reference is made to the following examples, which are
intended to illustrate the invention, but not limit the scope
thereof.
EXAMPLES
Example 1
Two Reagent Formulations Containing Different Reducing Agents for
an On-Board Control that can be Evaluated Optically
TABLE-US-00001 [0050] TABLE 1 Formulation for an OBC containing
glycine as a reducing agent that can be evaluated optically
Chemicals Source Concentration Sucrose Sigma 3.2 g/dl Mowiol 4/86
Clariant GmbH 1.3 g/dl Keltrol F Kelco 2.98 g/ml Glycine Sigma 1.35
g/dl HEPES.sup.1 Sigma 0.33 mg/ml polyethylene glycol PEG 3,350
Sigma 1.33 g/dl bovine serum albumin Sigma 0.4 g/dl mega 8 Sigma
0.67 mg/ml Resazurin Riedel de Haen 0.96 mg/ml .sup.1HEPES:
[4-(2-hydroxyethyl)-piperazino]-ethane sulfonic acid
[0051] The substances listed in Table 1 are mixed homogeneously and
adjusted to a pH of 7.4 with NaOH. The reaction mass obtained in
this manner was coated onto a test strip that can be measured by
reflection photometry as a reagent tape of 20 mm width and ca. 10
.mu.M thickness.
TABLE-US-00002 TABLE 2 Formulation for an OBC containing glucose as
a reducing agent that can be evaluated optically pH of the
formulation 7.5 9 10 11 Chemical Amount weighed out in g
polyvinylpyrrolidone 7.09 7.09 7.09 7.09 solution.sup.1 glucose 1.2
1.2 1.2 1.2 HEPES.sup.2 1.25 -- -- -- CHES.sup.3 -- 1.04 1.04 --
CAPS.sup.4 -- -- -- 1.11 bidistilled water 7.72 7.93 7.93 7.86
Keltrol F solution.sup.5 17.04 17.04 17.04 17.04 BM propiofan 70 D
5.69 5.69 5.69 5.69 titanium dioxide slurry.sup.6 55.32 55.32 55.32
55.32 resazurin 0.28 0.28 0.28 0.27 hexanol 0.17 0.17 0.17 0.17
methoxy-2-propanol 4.25 4.25 4.25 4.25 .sup.140 g/l
polyvinylpyrrolidone (PVP) is scattered into water and restirred
for about 30 min. .sup.2HEPES:
[4-(2-hydroxyethyl)-piperazino]-ethanesulfonic acid. .sup.3CHES:
2-(cyclohexylamino)-ethanesulfonic acid. .sup.4CAPS:
3-(cyclohexylamino)-1-propanesulfonic acid. .sup.56.8 g/l Keltrol
is scattered into water while stirring and restirred for several
hours. In order to ensure a complete swelling, the preparation is
allowed to stand overnight at room temperature before further use.
.sup.615 g titanium dioxide is scattered into 38 ml water while
stirring and then homogenized in a dissolver stirrer at a high
stirring rate.
[0052] The pH of the reagent mass is in each case adjusted to the
stated value with sodium hydroxide solution.
[0053] The substances listed in Table 2 are mixed homogeneously.
The reaction mass obtained in this manner was coated onto a test
strip that can be measured by reflection photometry as a reagent
tape of 20 mm width and ca. 10 .mu.m thickness.
[0054] The reaction rate of the OBC reaction i.e., the conversion
of resazurin into resorufin which depends on the environmental
conditions can be adjusted by the pH. The more alkaline the pH the
more rapid is this conversion and hence the more sensitive is the
OBC.
Example 2
Mass Spectroscopic Determination of the Degradation Product after
Stress in the Reagent Formulation According to Table 1 from Example
1
[0055] In the case of test strips that are intended to be stored at
room temperature in closed cans provided with desiccant or
moisture-impermeable foils, the degradation reaction should not
occur or only to a very slight extent under these storage
conditions.
[0056] The on-board control should in particular detect the
following as incorrect storage: [0057] high temperatures in closed
cans/foils (e.g., during transport to the customer or behind a pane
of glass in sunlight); and [0058] humid storage (cans that have not
been properly closed after removing test strips or defective foil
packaging).
[0059] These requirements were simulated by the following stress
models: [0060] Storage for several weeks at 50.degree. C. in closed
cans. [0061] Storage for hours and several days at 50.degree. C.
and elevated (50%/75%) air humidity. [0062] Storage for several
days in an open or defective packaging under environmental
conditions of climate zone 4 (30.degree. C., 70% air humidity).
[0063] The mass spectrum of a stressed reagent formulation which
was prepared according to Table 1 in Example 1 shows that after
stress (6 hours at 50.degree. C. and 75% relative air humidity) a
few percent of the resazurin was converted into resorufin (cf. also
FIG. 1). The main peak of the resorufin spectrum (212.20 mass
units) is present in addition to the main peak of the mass spectrum
of resazurin at 228.17 mass units.
Example 3
Absorption Spectra of Resazurin and Resorufin (FIG. 2)
[0064] The absorption spectra shown in FIG. 2 of resazurin 1 (a
blue dye) and resorufin 2 (a red dye) show that in principle it is
possible to optically detect (e.g., visually or by reflection
photometry) the conversion of resazurin 1 into resorufin 2 which
occurs when a reagent formulation is stressed as shown in Example
2.
Example 4
Reflection Spectra of Unstressed and Stressed Test Strips
[0065] Test strips whose reagent films were prepared based on the
formulation of Table 1 from Example 1 were stressed for 0 hours
(unstressed; cf. curve 1 in FIG. 3), 6 hours (slightly stressed;
cf. curve 2 in FIG. 3) and 12 hours (strongly stressed; cf. curve 3
in FIG. 3) at 50.degree. C. and 75% air humidity. The change in the
corresponding reflectance spectra was measured (FIG. 3). With an
increase in stress, an increase in the reflectance at a wavelength
of 620 nm is seen which is associated with a decrease in the amount
of resazurin.
Example 5
Optical Detection of the Degradation Product Resorufin in Test
Strips that were Stressed to Different Extents
[0066] Test strips whose reagent films were prepared on the basis
of the formulation of Table 1 from Example 1 were stressed for
different periods at 50.degree. C. and 75% air humidity and then
measured in a simple reflection photometer whose LED operated with
light at a wavelength of 620 nm.
[0067] FIG. 4 shows the increase in the degradation product
(recognizable by the increase in reflectance R) over the stress
time t (in h).
[0068] The change in the test field can also be readily detected
visually i.e., by the user with the naked eye. The reagent zone of
the unstressed test strip which is firstly blue changes its colour
into pink as the stress period increases.
Example 6
Formulation for a PT Coagulation Test with Integrated OBC and
Electrochemical Detection
TABLE-US-00003 [0069] TABLE 3 Formulation for an amperometric
prothrombin time test with integrated OBC reagents Chemical Source
Concentration relipidated recombinant human Dade Behring, 72 ng/ml
thromboplastin (rhTF) Marburg Sucrose Sigma 3.2 g/dl mowiol 4/86
Clariant GmbH 1.3 g/dl Keltrol F Kelco 2.98 g/ml Glycine Sigma 1.35
g/dl Polybrene Sigma 10 .mu.g/ml HEPES.sup.1 Sigma 0.33 mg/ml
polyethylene glycol PEG Sigma 1.33 g/dl 3,350 bovine serum albumin
Sigma 0.4 g/dl mega 8 Sigma 0.67 mg/ml Resazurin Riedel de Haen
0.96 mg/ml Electrozym TH Roche Diagnostics 1.2 mg/ml .sup.1HEPES:
[4-(2-hydroxyethyl)-piperazino]-ethanesulfonic acid
[0070] The substances listed in Table 3 were homogeneously mixed
and adjusted with NaOH to a pH of 7.4. The reaction mass obtained
in this manner was coated in a width of 4 mm and a thickness of ca.
90 .mu.m (wet) or ca. 10 .mu.m (dry) on a test strip that is to be
measured amperometrically such that the whole area of the working
electrode was covered with reagent. An Ag/AgCl electrode which also
served as the counter-electrode was used as a reference
electrode.
Example 7
Electrochemical Detection of Resazurin and the Degradation Product
Resorufin in Test Strips that were Stressed to Different
Extents
[0071] The test strips prepared according to Example 6 were
stressed for different periods at 50.degree. C. and 75% air
humidity. Whole blood was then measured amperometrically with these
test strips.
[0072] The following potentials were applied in order to quantify
resazurin and resorufin:
-700 mV vs. Ag/AgCl for 3 seconds (so-called OBC prepare phase);
subsequently -100 mV vs. Ag/AgCl for 1.5 seconds (so-called OBC
test phase); subsequently +200 mV vs. Ag/AgCl for 90 seconds (for
the actual coagulation measurement).
[0073] In order to quantify the "OBC prepare" and "OBC test"
signals, the integrals under the current-time curves were
calculated. FIG. 5 shows how these integrals (referred to as
Q.sub.700 and Q.sub.100) change with an increasing stress of the
test strips.
[0074] Whereas there is a major change in the OBC signals, the
clotting time measured with stressed test strips is almost stable
up to a very long stress period (cf. Table 4).
TABLE-US-00004 TABLE 4 Effects of stress at 50.degree. C. and 75%
relative air humidity on the measurement of clotting time Stress
time (h) Clotting time (s) 0 12.78 0.33 12.38 0.66 12.72 1 13.14 2
12.52 4 13.53 6 13.26 12 12.78 24 13.16 36 14.00
[0075] This ensures that the OBC indicates improper storage of the
test strips before false clotting times are generated.
Example 8
Indication of the OBC with Electrochemical Detection when
Unpackaged Strips are Stored in Climate Zone IV
[0076] The greatest risk of damaging test strips whose packaging is
defective or has not been closed again is in the case of customers
that live in climate zone IV (hot and humid). For this climate zone
a humidity of 70% and a temperature of 30.degree. C. have been
described in the literature as "average climate conditions".
[0077] Table 5 shows that the OBC with electrochemical detection
indicates changes in the test strip due to storage under these
conditions.
[0078] Table 5 shows on the basis of the measured charge Q (in nAs)
versus the stress time t (in h) the change in the electrochemically
measurable signal during a 3 second measuring phase at -700 mV vs.
Ag/AgCl (Q.sub.700) and during a 1.5 second measuring phase at -100
mM vs. Ag/AgCl (Q.sub.100) for different sample materials (normal
blood (N) 1 and 2; blood from donors that had been treated with
Marcumar (M) 1 and 2).
TABLE-US-00005 TABLE 5 Effects of a stress at 30.degree. C. and 70%
relative air humidity on the electrochemical on-board control for
different sample materials Q.sub.100 (nAs) Q.sub.700 (nAs) t (h) N
1 N 2 M 1 M 2 N 1 N2 M 1 M 2 0 58.42 57.3 51.7 56.8 2663 2512 2580
2735 2 58.62 57.9 56.0 58.0 2779 2551 2619 2748 6 62 62.0 62.3 63.6
2673 2567 2568 2728 9 62.21 61.4 61.0 63.8 2644 2466 2535 2713 12
65.16 63.4 61.7 65.8 2637 2488 2480 2723 24 77.07 76.0 75.6 78.8
2608 2455 2485 2722 48 94.56 89.0 84.4 89.2 2622 2493 2526 2665 72
102.3 104.3 100.1 105.2 2615 2455 2545 2705 96 119 123.7 122.1
127.5 2575 2466 2479 2722 120 151 141.8 145.0 167.2 2491 2434 2467
2535
[0079] Thus "stressed" analytical elements can be identified on the
bases of measurement data which were obtained during the so-called
OBC prepare phase (cf. Example 7) as well as during the so-called
OBC test phase (cf. Example 7).
[0080] As shown in Table 6, the clotting times are also much more
stable under these conditions and hence stressed test strips can be
reliably detected by the OBC before false coagulation measurement
values would have possibly been generated.
TABLE-US-00006 TABLE 6 Effects of stress at 30.degree. C. and 70%
relative air humidity on the measurement of clotting time Stress
time (h) Clotting time (s) 0 11.66 2 11.94 6 12.28 9 12.14 12 12.27
24 11.82 48 11.88 72 11.66 96 11.63 120 11.67
Example 9
Glycine as a Specific Redox Partner for Resazurin
[0081] If test elements which contain the formulation from Example
6 are stored for 48 hours at 25.degree. C., the ratio of resazurin
to resorufin changes because the former is converted into the
latter due to the "OBC reaction". The following Table 7 shows that
the reaction rate of this reaction is influenced by the presence or
absence of glycine. An OBC is also possible in the absence of
glycine; however, the sensitivity of the OBC is considerably
increased by the presence of glycine.
TABLE-US-00007 TABLE 7 Relative amount of resorufin (%) in relation
to the total amount of resorufin and resazurin versus the stress
period Resorufin percentage (%) Stress Reagent Reagent period (h)
without glycine with glycine 0 5 10 0.5 6 16 1 7 21 3 8 27 6 8 41 9
10 52 18 12 72 24 14 89 48 24 89
[0082] It is noted that terms like "preferably", "commonly", and
"typically" are not utilized herein to limit the scope of the
claimed invention or to imply that certain features are critical,
essential, or even important to the structure or function of the
claimed invention. Rather, these terms are merely intended to
highlight alternative or additional features that may or may not be
utilized in a particular embodiment of the present invention.
[0083] For the purposes of describing and defining the present
invention it is noted that the term "substantially" is utilized
herein to represent the inherent degree of uncertainty that may be
attributed to any quantitative comparison, value, measurement, or
other representation. The term "substantially" is also utilized
herein to represent the degree by which a quantitative
representation may vary from a stated reference without resulting
in a change in the basic function of the subject matter at
issue.
[0084] Having described the invention in detail and by reference to
specific embodiments thereof, it will be apparent that
modifications and variations are possible without departing from
the scope of the invention defined in the appended claims. More
specifically, although some aspects of the present invention are
identified herein as preferred or particularly advantageous, it is
contemplated that the present invention is not necessarily limited
to these preferred aspects of the invention.
* * * * *